Numerous challenges arise in production and purification of water soluble polymeric materials, not the least of which is the separation of specific polymers from impurities arising from synthetic processes. Following certain synthetic processes, polymers having one or more chemically reactive end groups must be separated from similar molecules having either a greater or lesser number of such reactive end groups.
There exists a specific need for the production of water soluble polymers of high purity that can react to form a single chemical bond with other target molecules. A common difficulty in these processes is the presence of contaminant polymer molecules having more than one reactive termini, that can form bonds to more than one of the target molecules or which form bonds to more than one different molecular species. In certain other applications, multiple bonds are the desired outcome and the removal of material with only a single reactive termini would improve the performance of such multiply reactive polymers.
Pharmaceutical science has undergone a rapid expansion in the types of agents used in treatment of disease and/or other disorders. Protein drugs are produced and marketed for the treatment of several human disease states. Small molecules are designed and developed that specifically interact with receptor sites found on cells, tumors, organs and the like. The effective and efficient delivery of materials has become an increasingly important aspect of the drug development and utilization process. Water soluble polymers such as poly(ethylene glycol) polymers (PEG polymers) and the monovalent mono ether derivatives of poly(ethylene glycol) such as monomethoxy poly(ethylene glycol) or MPEG polymers are valuable agents in the delivery and formulation of an ever increasing number of drug products. The structure of simple linear PEG and MPEG are shown in Formula 1A and Formula 1B respectively.

In some applications, both ends of a PEG polymer are utilized, for example, to couple a radioactively labeled material via the intervening polymer chain to a small molecule or small peptide that specifically binds to a cell receptor in vivo. These applications require material that can be manipulated differently at the two (or more) termini present.
Other applications utilize a MPEG material to increase the effective molecular weight and decrease the rate of elimination of a peptide or protein drug once introduced in vivo. In such applications it is highly preferred to have only a single reactive end and therefore only a single chemical bond formed between the polymer and the protein or peptide.
While it is in such pharmaceutical applications that the current invention will likely find its greatest application, the general difficulty in purification of polymeric materials after chemical modifications that effect only their end groups makes it beneficial to start with as homogenous (pure) a polymer as possible whenever such materials are to be modified. Further, the reversible nature of the modifications of the current invention and the manner in which it can enable purification of polymers not normally capable of ready purification has great utility whenever the preparation of modified forms of a water soluble polymer material is undertaken, even if the original polymeric material was quite pure.
The use of poly(ethylene glycol), PEG, and its mono-substituted methyl ether, MPEG, to conjugate to a protein has become so commonly practiced that the term PEGylation has been adopted to describe such protein conjugation. A search of the US patent application database of published applications for the term PEGylation gave over 2900 citations. Six of these applications contained PEGylation in the title, suggesting that such modifications to proteins and other molecules has become routine. Roberts et al. provides a review entitled “Chemistry for peptide and protein PEGylation,” in Advanced Drug Delivery Reviews 54 (2002) 459-476. An example of the modification of a biologically active peptide can be found in Lee et al., “Synthesis, Characterization, and Pharmacokinetic Studies of PEGylated Glucagon-like Peptide-1,” Bioconjugate Chemistry 16, (2005) 377-382. Rosendahl et al. describes site-specific protein PEGylation in BioProcess International, April 2005, 52-62. There is a demand for water soluble polymers such as poly(ethylene glycol) and MPEG derivatives of the highest purity.
In the preparation of MPEG polymers, the monofunctional product is produced by initiating the polymerization of ethylene oxide with, for example, sodium methoxide. Methyl ethers of ethylene glycol or of diethylene glycol can also be used. It is known that the synthesis of MPEG often produces a product with significant contamination with PEG. This PEG arises due to the presence of water in the polymerization mixture. Further, it is known that the ethylene oxide utilized in the preparation of MPEG polymer usually contains trace amounts of water as an impurity. This water can lead to the formation of additional PEG contaminant in a MPEG synthesis, as discussed in U.S. Pat. No. 6,448,369 to Bentley et al. U.S. Pat. No. 6,448,369 describes an alternative to the purification of the polymer in the preparation of heterobifunctional PEG derivatives in which the impurity is reacted to a relatively inert form by chemical reaction with a blocking group such that the impurity will not react with target molecules. While representing a moderately pragmatic approach for the short term, the use of such mixtures and the necessity of their remaining inert throughout use as well as the need to ultimately separate PEGylated materials from unreacted PEG reagents makes this approach less than ideal. Pharmaceutical usages would not benefit from the presence of such polymeric impurities. U.S. Pat. No. 6,448,369 also refers to chromatographic methods of Zalipshky in Bioconjugate Chemistry, 6 (1995) 150-165 and U.S. Pat. Nos. 5,747,639 and 5,935,564 to Seely, as applicable to the purification of polymeric PEGylation materials, but characterized such methods as tedious and having little value for useful commercial production.
U.S. Pat. Nos. 5,747,639 and 5,935,564 relate to hydrophobic interaction chromatography, which requires the use of high salt concentrations, expensive specialty media, and tends to have low binding capacities for the types of media utilized. The highest loading capacity disclosed in these patents involves the application of 8 mg of a PEG based material for each ml of chromatographic medium used, typically 3 mg of PEG per ml of material.
U.S. Pat. No. 5,747,639 describes PEG and the production of symmetrical bi-functional PEG derivatives. Separations described involve removal of PEG and PEG derivatives having only one end of the PEG derivatized with the desired moiety while the other end is present as an unmodified hydroxyl group. No route for separating reacted from unreacted MPEG materials is provided, and there is no suggestion of a method for purification and recovery of MPEG for use in the preparation of other derivatives.
An ion exchange separation of PEG derivative is described by Zalipsky in “Synthesis of an End-Group Functionalized Polyethylene Glycol-Lipid Conjugate for Preparation of Polymer-Grafted Liposomes”, Bioconjugate Chem. 4, 296-299 (1993) wherein derivatives are prepared that possess a carboxylic acid group at one or both ends. The anionic forms of these derivatives allow separation of PEG, the homobifunctional PEG diacid and the mono-substituted PEG monoacid. MPEG materials are not described and the method is intended for PEG materials that are 4,000 Da or less in size.
U.S. Pat. No. 5,298,410 to Phillips teaches that the ion exchange methods of Zalipsky et al. (Journal of Bioactive and Compatible Polymers, Vol. 5, April 1990, pp. 227-231) fail when higher molecular weight PEGs are used. This patent discloses the preparation of an MPEG fraction free of PEG at a molecular weight of 5,000 Da. Although there is some suggestion that higher molecular weight MPEG can be purified by the disclosed reverse phase chromatography methods, no examples are given, and the maximum size of the material referred to is 15,000 Da. The methodology described involves preparation of a reversible derivative of MPEG and the contaminating PEG. Triphenyl methyl (or trityl) ether derivatives are used and the method employs expensive acetonitrile containing elutants and expensive reverse phase silica chromatographic media. This patent describes the ion exchange purification of a mono-acid derivative of PEG made with PEG 2,000. A similar PEG based purification of mono-carboxylate substituted PEG from PEG and the di-carboxylate form is described in U.S. Pat. No. 5,672,662 to Harris and Kozlowski.
An ion exchange separation of PEG based materials that proceeds based on the substitution of one end of the PEG with a DMT (dimethoxytrityl) ether group is described by Drioli et al. (Reactive & Functional Polymers 48 (2001) 119-128). The omission of the DMT group allows small scale purification of a PEG mono-succinate from a PEG di-succinate, but with noted difficulty and overlap of the products. The DMT group was a key in the more successful routes and was added first in sufficient quantity to avoid, at least statistically, the presence in the final mixture of any starting free PEG. A subsequent reaction with a large excess of succinic anhydride allows the preparation of the DMT-PEG derivative free from PEG-bis succinic acid. This approach is not applicable to the preparation of MPEG free of PEG and was not demonstrated with PEG above 6,000 Da.
An alternative approach can be taken in which PEG derivatives are prepared with only one end of the PEG functionalized for conjugation to protein (or other material), instead of using MPEG mono-functional derivatives. U.S. Patent Application 2005/054816 by McManus et al. states that the current methods of preparing activated PEGs, particularly monosubstituted activated PEGs, are unsatisfactory because of reliance on the use of expensive MPEG starting material, containing contaminant PEG diol. Conventional synthetic approaches to avoid diol formation are complicated and can still result in the formation of detectable amounts of byproducts.
While U.S. Patent Application 2005/054816 demonstrates the purification of MPEG materials substantially free of PEG, it does so only when the PEG starting material has been partially reacted. Mixtures formed from a PEG are essentially of a common average molecular weight and have a homogeneous distribution of molecular weights. Example 7 of U.S. Patent Application 2005/054816 describes the use of a typical MPEG of 20,000 Da that contains 6 wt % of PEG-diol having a molecular weight of about 40,000 Da. A covalently bonded carboxylic acid group is produced in a three step reaction yielding a mixture that is 6% PEG (40,000 Da)-dipropionic acid and MPEG (20,000 Da)-propionic acid (91%) and MPEG (20,000 Da) (3%). This is purified using a chromatographic process to separate the neutral PEG materials from the mono-acid and the diacid forms. A complex chromatographic system is employed for separation, which uses a limited amount of media to provide some measure of selectivity for the charged species formed. In the final example, the same media is used in both the pre-column and the column. Thus, the basis of separation seems to be the marginal difference in binding between the mono-substituted and di-substituted material. In each case it is noted that the pre-column contains both the di-substituted PEG product and the monosubstituted desired product without providing much quantitative data on the relative yields. FIG. 4D of this publication illustrates that material bound to the pre-column is 80% mono and only about 20% PEG di-acid.
U.S. Patent Application 2005/054816 teaches the alkylation or substitution of the PEG alcohol or anion thereof with a halide, vinyl, tosyl or mesyl group wherein an ether type bond is formed between the PEG oxygen and the functionalizing reagent.
Chromatographic methods known as sample displacement chromatography allow purification of peptides by reversed-phase chromatography in a sample displacement mode rather than a gradient elution mode, as described Hodges et al. (J. Chromatogr. 1988; 444,349-62), or in U.S. Pat. No. 6,576,134 and U.S. Pat. No. 6,245,238 to Agner.
In U.S. Patent Application 2004/0062746 to Martinez et al., a method is described to prepare PEG having one reactive end and the other end as a hydroxyl group, not as a blocked end group such as with an MPEG derivative. Reverse phase chromatography is used to separate mixtures of PEG materials differing in their end group compositions, but no use of ion exchange is taught.
Oudhoff et al. described electrophoretic characterization and analysis of PEG using multiply charged derivative compounds in “Characterization of polyethylene glycols and polypropylene glycols by capillary zone electrophoresis and micellar electrokinetic chromatography.” J Chromatogr A. 985, 479-91(2003). The additional charge introduced using such materials as described by Oudhoff et al., as compared to a simple monoacid derivative, demonstrates only a standard mass to charge ratio increase in electrophoretic mobility and provides a more rapid separation. While this shortens the time of the analysis, it is not a major improvement in the analytical separation of species. The capillary electrophoresis methodology described is only an analytical method, and has no preparative applications. MPEG materials were not assessed.
One approach to obtaining high purity MPEG has been to exercise extraordinary care in the synthesis of MPEG from ethylene oxide and a methoxide initiator. U.S. Pat. No. 6,455,639 to Yasukohchi follows this route of production directly from oxirane materials that are greater than 98% pure. Statements in this patent suggest it is not possible to achieve a purity of greater than 98% by industrial separation/purification processes such as fractional liquid chromatography.
There remains a continuing need for high purity MPEG with very low PEG content, as well as a method to produce such high purity MPEG.